It is believed that therapeutic antibodies (mAbs) at least offer important treatment options for many diseases like inflammatory, autoimmune or oncological disorders. In 2012 there were 40 FDA-approved mAbs on the US market against various targets in oncology and anti-inflammatory disorders with ˜38.5% share within the biologics market. Sales of ˜$24.6 billion manifest the role of therapeutic antibodies as highest earning category of all biologics (Aggarwal, 2009, Aggarwal, 2014).
For therapeutic antibodies different biological outcomes are determined by the interaction profiles with four classes of naturally occurring interaction partners: antigen, neonatal Fc-receptor (FcRn), Fc-receptors (FcγRs), and factors of the complement system (Chan and Carter, 2010). Several strategies have been reported to optimize antibodies that aim for additional or improved functions and specificities. (Beck et al, 2010). Within antibodies there are two structural features that can be addressed for engineering. First, the variable fragment (Fv) that mediates interaction with the antigen, second the constant fragment (Fc) that is involved in antibody recycling or mediates interactions with immune cells.
For different antigens (e.g. cytokines and growth factors) there are multiple mechanisms of action, e.g. blocking of soluble ligands thereby preventing the interaction to its corresponding receptor or blocking of the receptor itself. (Chan and Carter, 2010). A lot of effort has been invested on improving functions towards Fv-engineering that is often valued by increasing specificity and/or binding affinity to respective antigens (Beck et al, 2010). One strategy to elevate antibody efficacy is to enhance the Fv—antigen interaction by affinity maturation approaches. Herein the use of display technologies for screening molecule libraries allows isolation of variants that exhibit superior affinity.
The constant fragment (Fc) and its linked properties can be modulated with altered outcomes for immunity or antibody recycling. Prominent examples for altered Fc-mediated immune functions are enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) that have been addressed to enhance antibody efficacy and to reduce the dosages.
Further strategies have been explored including direct and indirect arming of antibodies or modulation of specificities within multivalent antibodies (Carter 2011).
One important aspect of Fc-function corresponds to its critical role in antibody recycling that determines the long serum half life of human immunoglobulin 1 (IgG1). After cellular absorption via fluid phase pinocytosis, the Fc-portion of an IgG1 interacts in a pH-dependent manner with the neonatal Fc-receptor (FcRn) that leads to antibody capture in the acidified endosome (Kuo and Aveson, 2011). From there, antibodies are recycled back to the circulation and therefore can be protected from intracellular catabolism. Different mutational Fc-species with enhanced FcRn binding affinity were generated and tested for increased recycling rates with up to 4-fold extended serum half-life in cynomolgus studies by substituting three amino acids (Dall'Acqua et al, 2006).
Although most antibodies demonstrate highly efficient antigen blocking, there are drawbacks that are not fully addressed in the development process of therapeutic antibodies:                In many therapeutic antibodies the antigen-binding sites bind to only one antigen molecule during the antibody's lifetime in plasma (Igawa et al, 2010).        The dosing and frequency of antibody injections depends on the antigen synthesis rate between two injections as antigens are usually produced continuously in vivo (Igawa et al, 2010).        High production costs & administration of large quantities of antibodies: Tocilicumab therapy: 8 mg/kg/month by i.v. (Maini et al, 2006), Adalimumab therapy: 40 mg every other week ($19,272/per person/per year (Schabert et al. 2013).        When antibodies bind soluble targets “antibody buffering” effects can occur in which the degradation of the antigen is prevented while being bound to the antibody. A pool of free antigen establishes from reversible dissociation of the antigen-antibody complex and therefore prolongs the in vivo persistence (O'Hear and Foote, 2005)/Finkelman et al, 1993)        
Considering these drawbacks of therapeutic antibodies, there is a need for more efficient molecules that produce therapeutic responses without high dosing and/or frequent administration (Chapparo-Riggers et al, 2012).
One possibility to achieve these goals is the specific engineering of the variable and optionally the constant region of a new or well established and approved antibody. One approach is to develop antibodies that exhibit pH-sensitive antigen binding. It has been shown that rational or combinatorial incorporation of histidines in the binding interfaces of antibodies and other proteins (Sarkar et al., 2002, Chaparro-Riggers et al., 2012, Ito et al., 1992, Igawa et al., 2010, Igawa et al., 2013, Murtaugh et al., 2011, Gera et al., 2012) can be commonly used to engineer pH-dependent binding. The basis for the pH-sensitive binding arises from the histidine's sensitivity to get protonated as a result of lowered pH-values in the microenvironment. More in detail, the histidines need to undergo a pKa-change upon binding in order to get protonated in a physiological pH-range (Murtaugh et al., 2011). Protonation of histidine side chains in binding-interfaces can alter electrostatic interactions or may induce conformational changes that lead to pH-dependent differences in binding affinity (Gera et al., 2012). Balanced electrostatic and non-electrostatic components of the binding equilibrium determine the sensitivity of binding (Murtaugh et al., 2011).
Incorporation of pH-sensitivity into the antigen binding site can increase the number of antigen-binding cycles. Herein, pH-dependent antibodies bind with similar high or reduced sufficient affinity to their antigens at plasma pH (pH 7.4) and show decreased binding at acidic pH (pH 6) (Chaparro-Riggers et al., 2012, Igawa et al, 2010) resulting in a faster and increased dissociation of the antibody from its antigen binding site within the acidic endosome, thereby enabling recycling back to the plasma and reducing antigen-mediated clearance.
During the FcRn-mediated recycling (FIG. 1a) of conventional antibodies that bind soluble targets, the antibody-antigen complex is recycled back to the extracellular space through the endosomal trafficking pathway (Roopenian and Akilesh, 2007). In contrast, pH-sensitive antibodies (FIG. 1b) release their antigen from the antibody-antigen complex during the endosomal acidification (pH<6.5) (Roopenian and Akilesh, 2007). As a result the free antibody gets recycled to the circulation whereas the antigen enters the degradative pathway (Chaparro-Riggers et al., 2012, Igawa et al, 2010).
The pH-sensitive binding therefore enables the antibody to interact with another antigen and allows the neutralization of multiple antigen molecules per antibody molecule. PH-sensitivity can also increase the half-life of antibodies that address membrane associated targets and internalize and degrade upon e.g. receptor binding. Herein pH-sensitivity can lead to increased half-life, when the antibody gets released during the endosomal acidification.
Several different strategies were published that aim for the engineering of pH-switches in proteins. Histidine (His) scanning by which every single amino acid residue (e.g. within the CDR regions) is mutated to His allows the characterization of single substitution variants and identification of effective mutations. Creation of new variants by combining these substitutions can result in enhanced pH-dependent binding (Murtaugh et al., 2011, Chaparro-Riggers et al., 2012, Igawa et al, 2010). Identification of residues that may contribute to pH-sensitivity upon replacement with histidines by structure-based modeling can help to minimize effort & time that is needed during the histidine scanning approach. Crystal structures are required in order to have a precise idea of residues that are critical for binding and the rational design of pH-switches (Sarkar et al., 2002). Combinatorial histidine scanning library approaches require in vitro screening technologies (e.g. phage display or yeast display) to isolate pH-sensitive variants from a large molecule library. Murtaugh and colleagues designed a llama VHH antibody library by using oligonucleotide-directed mutagenesis thereby allowing every residue within the binding interface to sample both histidine residues and wild-type residues of the parental VHH antibody. Towards screening of a M13-phage display library (diversity ˜1012) isolated variants showed KDs between 35-91 nM at pH 7.4 and a ˜104 fold decrease in binding affinity at pH 5.4 (Murtaugh et al. 2011).
Since the recycled free antibody is capable of binding to another antigen, pH-dependent antigen binding would enable a single antibody molecule to repeatedly bind to multiple antigens, in contrast to the conventional approach in which a single antibody can bind to antigen only once.
Therefore, it is a general need to make available antibodies which are more effective with respect to their plasma and serum concentration, which can be achieved by installing pH sensitivity with respect to different cellular action sites of the therapeutic antibody.